Reference to any prior art in this specification is not and should not be taken as an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Double-stranded DNA (dsDNA) can be converted to a single-stranded DNA (ssDNA) by separating the strands or by removing one strand of the duplex. Strands of the duplex can be separated by thermal or chemical means of disrupting interstrand bonds. Removal of one strand permits recovery of the desired strand and elimination of its complement e.g. Nikiforov et al. (U.S. Pat. No. 5,518,900), who described modifying one of two primers used for amplification by incorporation of phosphorothiate nucleotide derivatives in the 5′ end of the modified primer, rendering it resistant to exonuclease digestion. After amplifying target sequences using the polymerase chain reaction (PCR), the dsDNA is subjected to exonuclease digestion. The unprotected strand is preferentially digested by a 5′ to 3′ exonuclease, leaving a single-stranded product consisting of the other strand. Similar strategies have used exonuclease-resistant branched primers (Shchepinov et al, Nuc. Acids. Res. 25:4447-4454 1997) or 5′ phosphate-bearing substrate preference of Lambda exonuclease (Higuchi et al, Nucl. Acids Res. 25:5685, 1989).
Asymmetric PCR (Gyllensten and Erlich, Proc. Natl. Acad. Sci. USA 85:7652-7656 1998; U.S. Pat. No. 5,066,584) generates ssDNA during thermocycling by employing an imbalanced primer pair concentration such that one primer is at a limiting concentration. This favours ssDNA product primed by the primer in excess. This approach has the problem of being inherently limited in processivity, since, by necessity, one primer is used at a relatively low concentration.
Competitor primer asymmetric PCR (Gillespie, 1997; U.S. patent application Ser. No. 08/628,417) employs the separate addition of competitor primer following PCR thermocycling and prior to further thermocycling to generate ssDNA. As such, this method requires excessive handling which is undesirable particularly in a diagnostic context due to increased risk of contamination, user error and processing time and cost.
Kaltenboeck et al, Biotechniques 12:164-171, 1992 described a method of producing ssDNA by initially performing a PCR to generate dsDNA, followed by a separate reaction using the product of the first PCR as a template for a second linear amplification employing one primer. Again, this method requires excessive handling.
Solid phase matrices have been labeled with PCR products using symmetric PCR or asymmetric PCR where one primer is conjugated to a solid surface or via a ‘bridge’ PCR where forward and reverse primers are directly conjugated to a solid surface. Each of these approaches is relatively inefficient due to kinetic constraints (low effective substrate concentrations with or without competitive inhibitory effects). If required, dsDNA products conjugated to a solid phase can be converted relatively simply to ssDNA products conjugated to the solid phase by chemical or thermal denaturation.
U.S. Pat. No. 6,277,604 describes the use of an immobilized and two aqueous phase primers in asymmetric PCR. At least one of the aqueous primers is provided in limiting concentration to facilitate the immobilized primer priming an extension event. However, this can lead to inefficiencies.
There is a clear need to develop more efficient methods for generating specific single stranded nucleic acid molecules and to label solid supports.